The plasmid pGSA1285 was first modified by substituting its GUS sequence with the Chalcone synthase intron fragment from vector pFGC5941 to get the plant silencing expression vector that contained Kanamycin resistance...The plasmid pGSA1285 was first modified by substituting its GUS sequence with the Chalcone synthase intron fragment from vector pFGC5941 to get the plant silencing expression vector that contained Kanamycin resistance site and was named as pGSA2285. Using PCR-based amplification, two different restriction sites at both ends of tobacco Bax inhibitor-1 （NtBI-I） gene were created, respectively, which made the construction of ihpRNA gene silencing vector more efficiently. Then, NtBI-1 genes were inserted into Multiple Cloning Site （MCS） of pGSA2285 respectively to form Bax inhibitor-1 ihpRNA gene silencing vector, named as pGSA4285, containing sense and anti-sense BI-1 sequence which was spliced by chalcone synthase intron. Combined PCR identification and enzyme restriction analyses, the results showed that Bax inhibitor-1 ihpRNA gene silencing vector had been constructed and transferred into Agrobacterium tumefaciens EHA105 successfully, which laid a foundation for the further study on the function of BI-1 in plant PCD regulation.展开更多
EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was...EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.展开更多
基金Supported by National Natural Science Foundation of China (30500352)China Agricultural University URP Project
文摘The plasmid pGSA1285 was first modified by substituting its GUS sequence with the Chalcone synthase intron fragment from vector pFGC5941 to get the plant silencing expression vector that contained Kanamycin resistance site and was named as pGSA2285. Using PCR-based amplification, two different restriction sites at both ends of tobacco Bax inhibitor-1 （NtBI-I） gene were created, respectively, which made the construction of ihpRNA gene silencing vector more efficiently. Then, NtBI-1 genes were inserted into Multiple Cloning Site （MCS） of pGSA2285 respectively to form Bax inhibitor-1 ihpRNA gene silencing vector, named as pGSA4285, containing sense and anti-sense BI-1 sequence which was spliced by chalcone synthase intron. Combined PCR identification and enzyme restriction analyses, the results showed that Bax inhibitor-1 ihpRNA gene silencing vector had been constructed and transferred into Agrobacterium tumefaciens EHA105 successfully, which laid a foundation for the further study on the function of BI-1 in plant PCD regulation.
基金Supported by the Natural Science Foundation of Heilongjiang Province(LH2021C032)。
文摘EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.
